Peptide Agonist

The present teaching relates to peptides that are capable of regulating the trafficking of leukocytes. This has applications in the treatment and/or prophylaxis of conditions associated with leukocyte migration, including inflammatory diseases and/or musculoskeletal (MSK) loss and/or damage.

Targeting inflammation in chronic disease represents a huge unmet need in clinical practice. This has been recognized for many years, however efforts to develop anti-inflammatory drugs have largely targeted molecules that promote inflammation. It is now known that many of these agents are functionally redundant, and 30 years of effort has yielded very few efficacious drugs. Moreover, even when inflammation is well controlled, bone damage can continue, and reparative normal bone formation remains poor. Thus, new strategies are required to develop effective treatments of inflammation and/or of MSK loss and/or damage.

Exaggerated or prolonged inflammation and leukocyte trafficking and retention in affected tissues are the hallmark of many common disorders, including inflammation such as immune mediated inflammatory diseases (IMIDs), that can lead to disability and death. This means that restitution of control over the inflammatory response represents a realistic target for therapeutic intervention in such diseases. Despite this, the in-built regulators of immune cell trafficking remain poorly understood.

Studies carried out by Chimen et al in2015, 21 (5), 467-475, have identified a peptide released following the stimulation of B cells with adiponectin. This peptide is capable of regulating T-cell trafficking in inflammatory conditions and is known as the peptide inhibitor of trans-endothelial migration, or PEPITEM (SEQ ID NO. 1).

PEPITEM comprises 14-amino acids and is a proteolytically derived peptide that spans residues 28 to 41 of its parent protein, 14-3-3 zeta/delta (14-3-3ζδ), which comprises 245 amino acids and is a product of the YWHAZ gene. 14-3-3 proteins contain seven isoforms and are known to be expressed in all eukaryotic cells. The family is capable of binding to a multitude of signalling molecules, such as kinases, phosphatases and transmembrane receptors, highlighting their ability to influence and regulate processes such as the cell cycle (see Bridges, D. and Moorhead, G. B.,2005, 296, re10; and Wilker, E. and Yaffe, M. B.,2004, 37, 633-42).

PEPITEM was first described as being secreted from B cells and inhibiting T-cell transmigration. Studies carried out using an in vitro model of transmigration (Chimen et al, supra.) suggest that the secretion of PEPITEM from B-cells following their treatment with adiponectin could regulate the transmigration of peripheral blood lymphocytes (PBLs) through human umbilical vein endothelial cells (HUVECs) stimulated with TNF-α and IFN-γ. Chimen et al. report that PEPITEM has efficacy in murine models of Sjogren's disease, uveitis (ocular disease), septicaemia and ischaemia/reperfusion injury. Using a biotin-conjugate on the N-terminal of PEPITEM, Chimen et al. identified cadherin-15 (CDH-15) as the endothelial receptor of PEPITEM. CDH-15 is a transmembrane glycoprotein which functions as a Cadependent cell adhesion molecule.

In WO 2013/104928 (The University of Birmingham), it is described that PEPITEM supports a homeostatic pathway which works to limit the magnitude of the inflammatory response irrespective of how it is initiated. The efficacy of PEPITEM across such a broad spectrum of IMIDs makes a strong case for the translation potential of the pathway.

In WO 2018/165218 (Allysta Pharmaceuticals, Inc.), PEPITEM is reported to be useful in the treatment of dry eye and ocular diseases of inflammation.

Abnormalities in the bones or joints of individuals underpin pathology in musculoskeletal (MSK) diseases, such as inflammatory arthritidies, osteoporosis, cancer-induced bone disease, Paget's disease of bone and the rare groups of metabolic bone diseases; where patients suffer permanent loss of function and pain. Moreover, patients with rheumatoid arthritis, psoriatic arthritis and/or osteoarthritis are likely to suffer from inflammation induced bone damage resulting in the need for joint replacement surgery. Sedentary activity where the bones are not actively loaded, such as prolonged bed-rest (>5 days) due to, for example, disease/surgery and hospitalisation or space travel, leads to loss in bone mass.

Maintenance of bone integrity is a key medical challenge, especially in ageing populations. MSK diseases affect >10 million people in the UK, costing the NHS ˜£4.7 billion per year and accounting for over 30 million working days lost per annum (Musculoskeletal data Advisory group response to the Government's mandate to NHS England 2017/18). Existing therapies focus on reducing joint pain and/or slowing the rate of bone damage, whilst therapies inducing bone repair and limiting bone loss are often ignored.

Bone growth and repair is dependent predominantly on the activities of osteoblast and osteoclast cells (see Raggatt, L. J., J. Biol. Chem., 2010, 285, 25103-25108). Osteoblast cells are the major cellular component of bone and almost the entire bone matrix in a mammal is mineralised by osteoblasts. Osteoblasts synthesise and mineralise bone during both bone formation and bone remodelling. In contrast, osteoclasts break down and restructure bone tissue by producing enzymes that dissolve the collagen, calcium and phosphorus of the bone. Currently, anti-resorptive bisphosphonates are typically used to treat osteoporosis, which inhibit bone resorption by promoting apoptosis of osteoclasts. However, long-term use is associated with increased incidence of micro-fractures and atypical femur fractures, suggesting that these drugs may hinder normal bone remodelling and repair (see, for example, Haworth, A. E. and Webb, J. Br. J. Radiol., 2002, 85 (1018), 1333-1342). Newer drugs on the market include anti-RANKL antibody (denosumab); an src kinase inhibitor (saracatinib); and a cathespin K inhibitor (odanacatib), which was discontinued in 2016 due to increased risk of stroke (see Hanley, D. A. et al., Int. J. Clin. Pract., 2012, 66 (12), 1139-1146; Danson, S. et al., J. Bone Oncol., 2019, 19, 100261; Bromme, D. and Lecaille, F., Expert Opin. Investig. Drugs, 2009, 18 (5), 585-600). These agents help to reduce the rate of bone damage by altering the activity of osteoclasts and preventing bone resorption. However, none affect osteoblasts—the cells known to induce bone formation.

Methods of reducing bone loss and/or stimulating bone production by controlling the balance between osteoclast and osteoblast activity are likely to be useful in the treatment of MSK diseases and/or damage, including any disorder of accelerated bone loss or impaired bone remodelling, such as cancer-induced bone disease, Paget's disease of bone and the rare groups of metabolic bone diseases, or diseases associated with inflammation (e.g RA, OA). Agents that stimulate bone formation, i.e. which stimulate osteoblast activity, are likely to be particularly effective in such treatment, since bone formation and mineralisation would not be limited by the natural, potentially under-active activity of osteoblasts.

In U.S. 62/912,439 (The University of Birmingham), it is described that PEPITEM is effective in reducing bone loss and/or stimulating bone production when administered to a patient and/or bone cells in effective amounts.

In view of the above, PEPITEM has the potential to treat both inflammation and MSK damage simultaneously, providing an advantage over other therapies where either inflammation or MSK damage is treated by a drug, thus patients require two different drugs, one per condition.

Although PEPITEM has been identified as efficacious against T-cell trafficking via interactions with CDH-15 and in reducing bone loss and/or stimulating bone growth, the core functional motif present in the peptide that mediates its function has not been reported. A fuller understanding of the core functional motif of PEPITEM is required to take full advantage of its potential as a regulator of leukocyte trafficking and an inhibitor of bone loss/promotor of bone growth, and to take advantage of it being easier and cheaper to manufacture than peptides of longer lengths (such as PEPITEM). The present invention aims to address this.

The inventors have found that peptides described herein are surprisingly effective regulators of leukocyte trafficking and stimulators of osteoblast activity. The peptides comprise no fewer than 3 and no more than 7 amino acids. Consequently, they are relatively short peptides and may be advantageously easier and cheaper to manufacture than peptides of longer lengths, such as PEPITEM. Some of the peptides of the disclosure have surprising additional advantages over PEPITEM as a regulator of leukocyte trafficking, such as increased efficacy, i.e. smaller half maximal inhibitory concentration (IC) values, longer half-lives (T) in plasma, better amenability to transport by peptide transporters, better solubility and/or, in some instances, better stability.

The skilled person is aware that any reference to an aspect of the current disclosure may include any embodiment of that aspect. For example, any reference to the first aspect may include the first aspect and any embodiments of the first aspect.

Viewed from a first aspect, there is provided a peptide of 3-7 amino acid residues for use as a medicament wherein the peptide comprises formula (I) and/or formula (II); wherein formula (I) is:

Viewed from a second aspect, there is provided the peptide of the first aspect for use in regulating leukocyte migration.

Viewed from a third aspect, there is provided the peptide of the first aspect for use in the treatment and/or prophylaxis of inflammation and/or musculoskeletal (MSK) loss and/or damage.

Viewed from a fourth aspect, there is provided a method of reducing bone loss and/or stimulating bone production, the method comprising administering an effective amount of the peptide of the first aspect ex vivo directly to bone cells and/or their precursors.

Viewed from a fifth aspect, there is provided a peptide of 3-7 amino acid residues as defined in the first aspect, wherein the peptide is not of sequence SVT, i.e. Ser-Val-Thr (SEQ ID NO. 2).

Viewed from a sixth aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the first or fourth aspect and a pharmaceutically acceptable excipient.

The peptides of the disclosure comprise no fewer than 3 and no more than 7 amino acids and in certain embodiments are surprisingly effective regulators of leukocyte trafficking and inhibitors of bone loss/promotors of bone growth. They may be advantageously easier and cheaper to manufacture than peptides of longer lengths, such as PEPITEM, and some may exhibit increased efficacy, i.e. smaller ICvalues, longer Tvalues in plasma, better amenability to transport by peptide transporters, better solubility and/or in some cases better stability than PEPITEM.

In the discussion that follows, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds disclosed herein, is generally based on the rules of the IUPAC organisation for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if a rule of the IUPAC organisation is contrary to a definition provided herein, the definition herein is to prevail.

The term “comprising” or variants thereof is understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term “consisting” or variants thereof is understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ±5% of the value specified. For example, if the level of inhibition of leukocyte migration is such that migration is reduced by at least about 30%, a reduction of 31.5% and a reduction of 28.5% is included.

The term “alkyl” is well known in the art and defines univalent groups derived from alkanes by removal of a hydrogen atom from any carbon atom, wherein the term “alkane” is intended to define cyclic or acyclic branched or unbranched hydrocarbons having the general formula CH, wherein n is an integer ≥1. A Calkyl group is an alkyl group having from one to six carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, and isohexyl.

As is known in the art, 2-amino-2-methylpropanoic acid (also known as α-aminoisobutyric acid (AIB), α-methylalanine or 2-methylalanine) is a non-proteinogenic amino acid with the structural formula NHC(CH)COOH.

As is known in the art, pyroglutamic acid (pE) is a natural amino acid derivative of glutamine or glutamic acid, wherein the N-terminal amino group attacks the carbonyl group of the side chain in a nucleophilic substitution reaction, in which the side-chain amino group of glutamine or side-chain hydroxy group of glutamic acid is displaced, and a lactam forms.

The term “enantiomer” defines one of a pair of molecular entities that are mirror images of each other and non-superimposable, i.e. cannot be brought into coincidence by translation and rigid rotation transformations. Enantiomers are chiral molecules, i.e. are distinguishable from their mirror image.

The term “half maximal inhibitory concentration value” or “ICvalue” is used herein to refer to the amount of peptide required to inhibit T-cell migration by 50% of the maximum inhibition observed.

The term “half-life” or “T” is used to define the time taken for the concentration of the peptide in the blood or plasma to be reduced by 50%.

The term “solvate” is used herein to refer to a complex comprising a solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a mono-hydrate, di-hydrate, tri-hydrate etc, depending on the number of water molecules present per molecule of substrate.

The term “isotope” is used herein to define a variant of a particular chemical element, in which the nucleus necessarily has the same atomic number but has a different mass number owing to it possessing a different number of neutrons.

The term “treatment” defines the therapeutic treatment of a human or non-human animal, in order to impede or reduce or halt the rate of the progress of the condition, or to ameliorate or cure the condition. Prophylaxis of the condition as a result of treatment is also included. References to prophylaxis are intended herein not to require complete prevention of a condition: its development may instead be hindered through treatment in accordance with the invention. Typically, treatment is not prophylactic, and the compound or composition is administered to a patient having a diagnosed or suspected condition. By an “effective amount” herein defines an amount of the peptide or composition disclosed herein that is sufficient to impede the noted diseases and thus produces the desired therapeutic or inhibitory effect. The skilled person is aware that the effective amount of peptide is not restricted to amounts that lead to overall improvement of a condition. Rather, the effective amount includes amounts that reduce the rate of deterioration of a condition. The skilled person is further aware that an effective amount is likely to vary with the particular compound disclosed herein, the subject and the administration procedure used. It is within the means and capacity of the skilled person to identify the effective amount of the compounds and compositions via routine work and experimentation. Typically, the effective amount may lie within a range of 1 mg/Kg to 100 mg/Kg.

The term “prodrug” is used herein to refer to a compound which acts as a drug precursor and which, upon administration to a subject, undergoes conversion by metabolic or other chemical processes to yield a peptide disclosed herein.

The term “pharmaceutically acceptable excipient” defines substances other than a pharmacologically active drug or prodrug, which are included in a pharmaceutical product.

The term “intrathecal administration” defines administration of a compound by injection into the spinal canal, or into the subarachnoid space.

The term “intraosseous administration” defines administration of a compound by injection into the bone marrow.

The term “intravenous administration” defines administration of a compound by injection into a vein or veins.

The term “intramuscular administration” defines administration of a compound by injection into a muscle.

The term “subcutaneous administration” defines administration of a compound by injection into the subcutis, i.e. the layer of skin directly below the dermis and epidermis.

The term “oral administration” defines administration of a compound through the mouth, wherein the compound is typically in the form of a tablet or capsule.

“Prolonged bed rest” is used herein to refer to bed rest for a period of time ranging from several days to several months. The skilled person is aware that a patient is not necessarily immobile for the entirety of the period, or confined to bed because of a health impairment that physically prevents them from leaving bed. However, the patient is necessarily in bed for the majority of the period.

The term “biocompatible” is used herein to refer to a material that is not harmful or toxic to living tissue.

The peptide chains of all formulae herein, such as formulae (I) and (II) (Z—X—Zand Z-G-X), are written from the N-terminus to the C-terminus.